S103: NATURAL AND AGE-RELATED VARIATION IN CIRCULATING HUMAN HEMATOPOIETIC STEM CELLS
Authors: Nili Furer, Nimrod Rappoport, Amos Tanay, Liran Shlush
Journal: HemaSphere (2023)

Topic: 23. Hematopoiesis, stem cells and microenvironment Background: Interrogation of complete blood counts from large cohorts of age-diverse healthy individuals exposes large inter-individual heterogeneity across all age decimals, as well as specific age-related patterns, such as the development of high RDW macrocytic anemia and a reduction in absolute lymphocyte counts. The mechanisms underlying this normal variation remain unclear. Aims: To deepen our understanding of natural and age-related variation in blood counts, by providing a detailed description of inter-individual heterogeneity in HSPC compositions and transcriptional programs in a large cohort of healthy (normal CBC) individuals, and how these correlate with age, sex, blood count parameters, and presence of age-related clonal hematopoiesis. Methods: We performed scRNAseq on CD34+ circulating HSPCs from fresh PB samples of 99 healthy individuals aged 25-95 years. All sample donors were considered healthy, their CBCs were within normal range, and they were not known to have any ARCH-defining mutations prior to sampling. We used genotype-based multiplexing of samples, relying on common SNPs we identified in the 3' UTR of HSPC RNA and their targeted DNA sequencing, to reduce batch bias and robustly remove doublets. We collected longitudinal CBCs from all individuals in our cohort up to 5 years prior to sampling, and performed deep targeted somatic mutation analysis to identify cases of CH. Following quality control and filtering, we retained 360,000 single cell profiles with which we constructed a metacell manifold model of circulating HSPCs. Results: The magnitude of our cohort allowed us to characterize rare populations of HSPCs that could not be studied in depth previously, such as circulating HLF/AVP positive HSCs, known to have extensive self-renewal capacity, as well as a common-NK/T/DC progenitor which does not decline with age. Inter-individual heterogeneity in the frequency of specific cell states was highly correlated with certain blood indices, specifically linking CLP frequencies with %Lymphocytes, and MEP frequencies with hematocrit. Interestingly, Low CLP frequencies, a high RDW and the presence of CH, were all individually linked to each other. Compositional-controlled analysis of inter-individual transcriptional variation in HSPCs exposed a Lamin-A (LMNA) signature, which was also correlated with CH and a reduction in CLP frequencies, as well as an age-correlated S-phase signature observed in the late MEP trajectory. Aberrant synchronization in closing stem cell programs and opening erythroid programs in MEP differentiation was linked to both RBC and MCV, such that individuals who closed their stem cell program too early had higher MCV and lower RBC values and vice-versa. Finally, as proof of concept, we introduce new methodologies for analyzing MDS/AML cases based on our normal reference atlas. All data can be observed herein https://tanaylab.weizmann.ac.il/MCV/blood_aging/ Summary/Conclusion: We portray a comprehensive map of circulating human HSPCs in an age-diverse, large, healthy cohort. While HSPC transcriptional states are generally conserved across individuals, we detect remarkable inter-individual variation in their internal distribution and regulation, and link these to various clinical parameters, as well as the presence of pre-leukemic mutations. Case studies of patients with early hematologic malignancies show highly abnormal circulating HSPC state distributions, and multiple novel gene signatures, suggesting further diagnostic opportunities based on the healthy reference map provided.Keywords: Hematopoietic stem and progenitor cells, Peripheral blood, RNA-seq, Aging